The overall objective of this grant proposal is to further analyze the role of DPIV/CD26 in antigen specific T cell activation, with special emphasis on AIDS related immune dysfunction. The basis is provided by our characterization of the T cell accessory function of CD26. Furthermore, we have recently shown that HIV-1 Tat binds tightly to CD26 and exerts its immunosuppressive effect through this molecule. We now propose to determine the underlying molecular mechanism of the CD26 mediated T cell co-stimulation, and to analyze the role that HIV-1 Tat plays in the antigen specific immunosuppression seen in T cells of HIV-1 infected individuals. 1. We will test the hypothesis that CD26 interacts with a membrane-bound physiological ligand to achieve its accessory function. For this purpose we will produce CHO cells which express a high level of either human or murine CD26 on their cell surface and engineer chimeric CD26 molecules, in conjunction with CD4, CD8 or mutant leucine zipper peptide, respectively. These reagents will be used to screen for specific binding of CD26 to thymic epithelial cell lines and/or conventional APC and peripheral lymphocytes. If binding is achieved, the ligand will be identified either by blocking with known mAbs to various surface molecules, or by cDNA expression cloning. 2. We will test the hypothesis that immuno-incompetence seen in CD4+ T cells early after HIV-1 infection is due to a defect in CD26 mediated signal transduction; namely, we will analyze whether recombinant CD26 molecules reverse the antigen unresponsiveness, and whether HIV-1 Tat blocks the association of adenosine deaminase with CD26. 3. & 4. To test the in vivo significance of the DP IV/CD26 molecule for T cell maturation and activation and the HIV-1 Tat mediated suppression, we will generated chimeric mice which either lack CD26 selectively on lymphocytes or selectively express HIV-1 Tat on these cells. This will be achieved by the injection of blastocysts derived from RAG-2-deficient mice with embryonic stem cells containing a homozygous mutation of the CD26 gene or are transfected with the HIV-1 Tat gene under control of T cell specific regulatory elements. These experiments represent a collaboration with Dr. Fred Alt's group at Harvard Medical School. We will generate a mutant construct of the CD26 gene for the selection of homologous recombinants. ES cells will be transfected with this construct, and cells that have undergone homologous recombination on both chromosomes will be selected. The chimeric offspring form the RAG-2-deficient blastocyst complementation assay will be tested for T cell development and function. If our working hypothesis is correct, these two types of mice should have similar functional profiles. These specific aims represent a combination of basic research on the mechanism of CD26 mediated T cell signaling in vivo and in vitro, as well as a direct analysis of clinical samples, derived from HIV-1 infected individuals, to test the role of CD26 in the observed immunosuppression. They are carried out in collaboration with Dr. W.W. Bachovchin's group who determines the physico-chemical properties of CD26 in association with various binding proteins.